We received female BALB/cJ mice (Jackson Laboratory, Bar Harbor, ME, USA) at 8 weeks of age. The animals were kept in isolated ventilated cages (IVC-Racks; BioZone, Margate, UK) supplied with filtered air, in a 12-hr light/12-hr dark cycle. Specific pathogen-free hygienic status was approved by a health certificate according to the Federation of European Laboratory Animal Science Associations guidelines (Nicklas et al. 2002 (link)). Food and water were available ad libitum. Animals were 10–12 weeks of age with body weights between 19.6 and 23.1 g during the study. Each of the 22 experimental groups consisted of eight animals. Twenty groups were exposed to particles, and two groups served as control and sham exposed. Mice were anesthetized by intraperitoneal injection of a mixture of xylazine (4.1 mg/kg body weight) and ketamine (188.3 mg/kg body weight). The animals were then intubated by a nonsurgical technique (Brown et al. 1999 (link)). Using a bulb-headed cannula inserted 10 mm into the trachea, a suspension containing 5, 20, or 50 μg particles, respectively, in 50 μL pyrogene-free distilled water was instilled, followed by 100 μL air. (For supplementary measurements, we used 0.5 and 2 μg ufCP.) The suspension of poorly soluble particles was sonicated on ice for 1 min prior to instillation, using a SonoPlus HD70 (Bachofer, Berlin, Germany) at a moderate energy of 20 W. We favor the use of distilled water for suspension of particles because the salt content of phosphate-buffered saline (PBS) causes rapid particle aggregation comparable to the “salting-out” effect (Shaw 1992 ) and thus eliminates consistent instillation conditions. In our experience, the instillation of 50 μL distilled water did not cause any measurable stress effects such as the expression of heat shock protein hsp70/hsp1a (data not shown). Control animals were not instilled, and sham animals received 50 μL pure distilled water. Animals were treated humanely and with regard for alleviation of suffering; experimental protocols were reviewed and approved by the Bavarian Animal Research Authority (approval no. 211-2531-108/99).
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